Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3962-3966, June 1987 Applied Biology Stable transformation of soybean by electroporation and root formation from transformed callus (Glycine max/direct DNA transfer/protoplasts/organogenesis) PAUL CHRISTOU, JEAN E. MURPHY, AND WILLIAM F. SWAIN Agracetus, 8520 University Green, Middleton, WI 53562 Communicated by Oliver E. Nelson, Jr., February 25, 1987 (receivedfor review December 3, 1986) ABSTRACT Soybean protoplasts from a number of com- to +1550, respectively (11)], and the aminoglycoside 3'- mercially important cultivars have been genetically engineered phosphotransferase II (APHII; kanamycin kinase, EC by way of electroporation using chimeric genes coding for 2.7.1.95) coding region from TnS [nucleotides 1541-2517 resistance to the aminoglycoside antibiotics kanamycin and (12)]. The coding region deletes an AUG triplet present in the G418. Effective electroporation conditions were determined by TnS sequence upstream and out-of-frame with respect to the monitoring transient expression from aminoglycoside 3'-phos- initiator AUG (13). The junctions between the pBR322, photransferase II (APHII) expression plasmids. Electropora- nopaline synthase, and APHII fragments include portions of tion of protoplasts with a chimeric APHII gene and subsequent synthetic polylinkers. A restriction map of pCMC1021 is selection on media supplemented with kanamycin resulted in shown in Fig. 1. Plasmid DNA was prepared by the method the recovery of calli resistant to the antibiotic. Enzyme assays of Ish-Horowicz and Burke (14), was twice-banded by for APHII activity and Southern blot hybridization confirmed isopycnic centrifugation in CsCl/ethidium bromide gradi- the expression ofthe foreign DNA and its stable integration into ents, and was chromatographed on NACS 52 (Bethesda the soybean genome. Root formation was induced from trans- Research Laboratories). formed calli, and these roots maintained expression of the Protoplast Isolation. Four- to eight-millimeter (10-20 day) APHII gene. zygotic embryos were excised from greenhouse grown plants (cultivars Williams 82, Mandarin Ottawa, and Hardin and Soybean (Glycine max) is one of the world's most important also Glycine canescens, a nondomesticated relative of G. agronomic crops. Accordingly, a great deal of effort has been max) and were cut transversely as described by Lu et al. (15). directed towards its genetic improvement by both conven- The chopped embryos were plasmolyzed for 1 hr in a 13% tional breeding techniques and genetic engineering approach- mannitol/salt solution (16) and then were incubated in the es. Successful application of standard genetic engineering enzyme mixture described by Lu et al. (15) for 5 hr at room procedures to soybean has been limited by the lack of an temperature on a gyratory shaker at 20 rpm. Protoplasts were efficient transformation system and the inability to regener- released by squeezing the embryo slices against the flask wall ate transformed tissues. Oncogenic transformation of soy- with a sterile spatula. The digestion mixture was sieved bean by virulent Agrobacterium strains and the axenic through a 54-,um stainless steel screen, and the screen was culture of excised tumors on hormone-free media have been rinsed with 2 ml of a 9% mannitol/salt solution (16). The reported (1-5), but disarmed Ti vectors have not yet been filtrate was transferred to 15-ml conical tubes and was used successfully. Agrobacterium strains have been isolated centrifuged at 135 x g for 8 min. The supernatant was that show increased virulence toward soybean and other discarded, and the protoplasts were washed twice in the same plants that are not particularly susceptible to crown gall solution by resuspension and centrifugation. The pellet was infection, and "disarmed" hosts for Agrobacterium vector collected and suspended for a final wash in a 0.49 M systems have been developed from these strains (6). How- sucrose/salt solution. Aliquots of the protoplast suspension ever, even with these strains, the transformation frequency is were diluted, stained with fluorescein diacetate, and counted very low relative to that obtained in alternative plant hosts on a hemocytometer grid under UV illumination. Protoplasts such as tobacco and petunia. Electroporation has been shown were then diluted with protoplast medium to the desired to be an effective technique for the transformation of mam- density for electroporation. malian cells (7). Fromm et al. (8) have shown that with Electroporation. Soybean protoplasts were electroporated appropriate modification the technique is also applicable to at densities of2-4 x 106 per ml in Kao protoplast medium (17) plant protoplasts, and they have achieved the stable trans- supplemented with 40 mM NaCl. One-milliliter aliquots ofthe formation of maize tissue cultures by this method (9). These protoplast suspension were pipeted into 1.5-ml disposable results, together with our own experience with electropora- cuvettes and were chilled briefly in an ice water bath. An tion of tobacco, prompted us to investigate this method as a electric pulse was delivered to the suspension by way of means of achieving the transformation of soybean. platinum wire electrodes with 1-cm spacings. The electric pulse was supplied by a 490-AF capacitor (Sprague Power- lytic 36D, Marsh Electronics, Milwaukee, WI) charged to the MATERIALS AND METHODS desired voltage with a voltage-regulated power supply. The Plasmids. pCMC1021 was constructed from a fragment of capacitor circuit was similar to that described by Fromm et pBR322 [nucleotides 2522-4361 (10)] containing the replica- al. (8) with the addition of a switchable bleed-resistor. tion origin and the 8-lactamase gene, a chimeric gene con- Voltages were monitored with a voltmeter, and the capacitor sisting of 5' and 3' regulatory regions from the nopaline discharge was followed on an oscilloscope. Under these synthase gene ofpTiT37 [nucleotides -265 to +36 and + 1298 conditions and with the capacitor charged to an initial voltage of 375 V, the discharge was an exponential decay with a The publication costs of this article were defrayed in part by page charge half-time of approximately 45 msec. payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: APHII, aminoglycoside 3'-phosphotransferase II. 3962 Downloaded by guest on October 2, 2021 Applied Biology: Christou et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3963 activity by the method of Reiss et al. (18). The amount of enzyme present in each sample was estimated by comparison to a known amount of purified enzyme included on the same gel. Southern Blots. DNA was prepared from lyophilized tissue by the method of Taylor and Powell (19). DNA was digested with restriction endonucleases under the conditions recom- mended by the supplier (New England Biolabs), was resolved by electrophoresis on an 0.8% agarose gel, and was trans- ferred to nylon membranes (Biodyne membranes, Pall, Ir- vine, CA) as described by Southern (20). 32P-labeled RNA hybridization probes were synthesized in vitro using an SP6 transcription system (Promega Biotec, Madison, WI) and [a-32P]GTP (300 Ci/mmol; Amersham; 1 Ci = 37 GBq). The template for probe synthesis produced a runoff transcript corresponding to the minus strand ofthe coding sequence for APHII. Reaction conditions recommended by the supplier, using 100 ,uCi of radiolabeled GTP, were used. Incorporation FIG. 1. Restriction map of plasmid pCMC1021. Components of was typically 40-60%. Hybridization and washing conditions the nopaline synthase/APHII chimeric gene are indicated by the were as described by Church and Gilbert (21). Filters were boxed regions. The hatched region represents the APHII coding analyzed by autoradiography using X-Omat AR5 film (Ko- sequence, and the open-boxed regions of the APHII fragment dak) at -80'C with two intensifying screens (Cronex Light- indicate noncoding portions of TnS included in the fragment. Nos ning Plus, DuPont). Promoter and Nos Poly A designate the 5' and 3' control regions of the nopaline synthase gene, respectively, as described in Materials and Methods. The arrow labeled bla indicates the position and RESULTS AND DISCUSSION polarity of the 3-lactamase gene derived from pBR322. The size of the entire plasmid is approximately 3.4 kilobases. A requisite step in recovering a stably transformed soybean tissue from electroporation experiments was to obtain large numbers of viable protoplasts that could be regenerated to Culture of Protoplasts and Selection of Stable Transform- callus. Tissue source was found to be crucial in protoplast ants. Electroporated protoplasts were incubated at 0C for preparation. Root, leaf, stem, and hypocotyl were tested, but approximately 5 min following application of the electric each ofthese tissues was inferior to zygotic embryos in terms pulse and then diluted into 10 ml of Kao's medium (17) in ofboth yield and viability. We found the protoplast yield also Corning 75-cm2 tissue culture flasks. The flasks were incu- to be dependent on embryo size, the optimum size being 4-8 bated in the dark at room temperature without agitation for mm. Larger embryos resulted in fewer protoplasts, presum- 8 days, at which time 5 ml ofKao cell culture medium (17) was ably due to a loss of viability caused by the high starch added dropwise, and the flasks were exposed to low light content of more mature embryos. A modification of the (2000 lux, room temperature).